Transdermal Botulinum Toxin Compositions

ABSTRACT

Pharmaceutical compositions for transdermal administration of neurotoxins to a patient include a neurotoxin, such as a botulinum toxin, and an enhancing agent that facilitates absorption of the neurotoxin through the skin of the patient and does not eliminate the bioactivity associated with the neurotoxin. The pharmaceutical compositions are topically applied on a patient, and may be provided in a transdermal patch.

The present invention relates to pharmaceutical compositions containingneurotoxins. In particular, the present invention relates tocompositions containing clostridial neurotoxins, such as botulinumtoxin, for transdermal topical administration to patients.

BACKGROUND Botulinum Toxin

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped according to their morphology and functions. Theanaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of a commercially available botulinum toxintype A (purified neurotoxin complex)¹ is a LD₅₀ in mice (i.e. 1 unit).One unit of BOTOX® contains about 50 picograms (about 56 attomoles) ofbotulinum toxin type A complex. Interestingly, on a molar basis,botulinum toxin type A is about 1.8 billion times more lethal thandiphtheria, about 600 million times more lethal than sodium cyanide,about 30 million times more lethal than cobra toxin and about 12 milliontimes more lethal than cholera. Singh, Critical Aspects of BacterialProtein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited byB. R. Singh et al., Plenum Press, New York (1976) (where the stated LD₅₀of botulinum toxin type A of 0.3 ng equals 1 U is corrected for the factthat about 0.05 ng of BOTOX® equals 1 unit). One unit (U) of botulinumtoxin is defined as the LD₅₀ upon intraperitoneal injection into femaleSwiss Webster mice weighing 18 to 20 grams each. ¹ Available fromAllergan, Inc., of Irvine, Calif. under the tradename BOTOX® in 100 unitvials)

Seven botulinum neurotoxins have been characterized, these beingrespectively botulinum neurotoxin serotypes A, B, C₁, D, E, F and G eachof which is distinguished by neutralization with type-specificantibodies. The different serotypes of botulinum toxin can vary in theanimal species that they affect and in the severity and duration of theparalysis they evoke. Botulinum toxin apparently binds with highaffinity to cholinergic motor neurons, is translocated into the neuronand blocks the release of acetylcholine.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain, H chain, and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for tetanustoxin. The carboxyl end segment of the H chain, H_(C), appears to beimportant for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This step is thought to be mediated by the amino end segment ofthe H chain, H_(N), which triggers a conformational change of the toxinin response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra-endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxin(or at a minimum the light chain) then translocates through theendosomal membrane into the cytoplasm.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the heavy chain, Hchain, and the light chain, L chain. The entire toxic activity ofbotulinum and tetanus toxins is contained in the L chain of theholotoxin; the L chain is a zinc (Zn++) endopeptidase which selectivelycleaves proteins essential for recognition and docking ofneurotransmitter-containing vesicles with the cytoplasmic surface of theplasma membrane, and fusion of the vesicles with the plasma membrane.Tetanus neurotoxin, botulinum toxin types B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytoplasmic surface of the synaptic vesicle is removed asa result of any one of these cleavage events. Botulinum toxin serotype Aand E cleave SNAP-25. Botulinum toxin serotype C₁ was originally thoughtto cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Eachof the botulinum toxins specifically cleaves a different bond, exceptbotulinum toxin type B (and tetanus toxin) which cleave the same bond.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. A botulinum toxin type A complex (BOTOX®) has been approved bythe U.S. Food and Drug Administration for the treatment ofblepharospasm, strabismus and hemifacial spasm, cervical dystonia andtreatment of glabellar wrinkles. A type B botulinum toxin (MYOBLOC™) hasalso been approved by the FDA for the treatment of cervical dystonia.Non-type A botulinum toxin serotypes apparently have a lower potencyand/or a shorter duration of activity as compared to botulinum toxintype A. Clinical effects of peripheral intramuscular botulinum toxintype A are usually seen within a day or a few hours after injection. Thetypical duration of symptomatic relief from a single intramuscularinjection of botulinum toxin type A averages about three to four months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinum typesA and E both cleave the 25 kilodalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Apparently, a substrate for a botulinum toxin can be found in a varietyof different cell types. See e.g. Biochem, J 1; 339 (pt 1):159-65:1999,and Mov Disord, 10(3):376:1995 (pancreatic islet B cells contains atleast SNAP-25 and synaptobrevin).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 700 kD or 500 kD complex. Botulinum toxin type D isproduced as both 300 kD and 500 kD complexes. Finally, botulinum toxintypes E and F are produced as only approximately 300 kD complexes. Thecomplexes (i.e. molecular weight greater than about 150 kD) are believedto contain a non-toxin hemaglutinin protein and a non-toxin andnon-toxic nonhemaglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine (Habermann E., et al., Tetanus Toxin and Botulinum A andC Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse Brain, JNeurochem 51(2); 522-527:1988) CGRP, substance P and glutamate(Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks GlutamateExocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J.Biochem 165; 675-681:1897. Thus, when adequate concentrations are used,stimulus-evoked release of most neurotransmitters is blocked bybotulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterizationof Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9);1373-1412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin InhibitsNon-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons inCulture, Brain Research 360; 318-324:1985; Habermann E., Inhibition byTetanus and Botulinum A Toxin of the release of [ ³ H]Noradrenaline and[ ³ H]GABA From Rat Brain Homogenate, Experientia 44; 224-226:1988,Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Releaseand Uptake of Various Transmitters, as Studied with ParticulatePreparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's ArchPharmacol 316; 244-251:1981, and; Jankovic J. et al., Therapy WithBotulinum Toxin, Marcel Dekker, Inc., (1994), page 5.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can Aberecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Shantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Shantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56; 80-99:1992. Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample; purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Botulinum toxins and/or botulinum toxin complexes can be obtained fromAllergan Inc (Irvine, Calif.), Ipsen Beaufour (France), ElanPharmaceuticals (Ireland), List Biological Laboratories, Inc., Campbell,Calif.; the Centre for Applied Microbiology and Research, Porton Down,U.K.; Wako (Osaka, Japan), Metabiologics (Madison, Wis.) as well as fromSigma Chemicals of St Louis, Mo.

Though somewhat labile, pure botulinum toxin can be used to prepare apharmaceutical composition and like the botulinum toxin complexes, suchas the toxin type A complex, is susceptible to denaturation due tosurface denaturation, heat, and alkaline conditions. Inactivated toxinforms toxoid proteins which may be immunogenic. The resulting antibodiescan render a patient refractory to toxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependent, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin can be stabilized with astabilizing agent such as albumin and gelatin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown in a medium containingN-Z amine and yeast extract. The botulinum toxin type A complex ispurified from the culture solution by a series of acid precipitations toa crystalline complex consisting of the active high molecular weighttoxin protein and an associated hemagglutinin protein. The crystallinecomplex is re-dissolved in a solution containing saline and albumin andsterile filtered (0.2 microns) prior to vacuum-drying. The vacuum-driedproduct is stored in a freezer at or below −5° C. BOTOX® can bereconstituted with sterile, non-preserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX®, sterile normal saline without apreservative; (0.9% Sodium Chloride Injection), is used by drawing upthe proper amount of diluent in the appropriate size syringe. SinceBOTOX® may be denatured by bubbling or similar violent agitation, thediluent is gently injected into the vial. For sterility reasons BOTOX®is preferably administered within four hours after the vial is removedfrom the freezer and reconstituted. During these four hours,reconstituted BOTOX® can be stored in a refrigerator at about 2° C. toabout 8° C. Reconstituted, refrigerated BOTOX® has been reported toretain its potency for at least about four weeks. Dermatol Surg 1996January; 22(1):39-43.

It has been reported that botulinum toxin type A has been used inclinical settings as follows:

(1) about 75-125 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercihii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

(a) flexor digitorum profundus: 7.5 U to 30 U

(b) flexor digitorum sublimus: 7.5 U to 30 U

(c) flexor carpi ulnaris: 10 U to 40 U

(d) flexor carpi radialis: 15 U to 60 U

(e) biceps brachii; 50 U to 200 U. Each of the five indicated muscleshas been injected at the same treatment session, so that the patientreceives from 90 U to 360 U of upper limb flexor muscle BOTOX® byintramuscular injection at each treatment session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

Additionally, intramuscular botulinum toxin has been used in thetreatment of tremor in patients with Parkinson's disease, although ithas been reported that results have not been impressive. Marjama-Jyons,J., et al., Tremor-Predominant Parkinson's Disease, Drugs & Aging 16(4);273-278:2000.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and insome circumstances for as long as 27 months. The Laryngoscope109:1344-1346:1999. However, the usual duration of an intramuscularinjection of Botox® is typically about 3 to 4 months. The success ofbotulinum toxin type A to treat a variety of clinical conditions has ledto interest in other botulinum toxin serotypes. See e.g. Eur J Neurol1999 November; 6(Suppl 4):S3-S10.

In addition to having pharmacologic actions at the peripheral location,botulinum toxins may also have inhibitory effects in the central nervoussystem. Work by Weigand et al, Nauny-Schmiedeberg's Arch. Pharmacol1976; 292 161-1651 and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol.1974; 281, 47-56 showed that botulinum toxin is able to ascend to thespinal area by retrograde transport. As such, a botulinum toxin injectedat a peripheral location, for example intramuscularly, may be retrogradetransported to the spinal cord.

U.S. Pat. No. 5,989,545 discloses that a modified clostridial neurotoxinor fragment thereof preferably a botulinum toxin, chemically conjugatedor recombinantly fused to a particular targeting moiety can be used totreat pain by administration of the agent to the spinal cord.

Botulinum toxin is most frequently administered as a therapeutic agentby injecting a composition containing botulinum toxin into a patientusing a needle or syringe. However, other modes of administration havebeen considered for botulinum toxins as well as botulinum toxins coupledwith non-botulinum toxin receptor legends. Some modes of administrationinclude topical application of botulinum toxin (e.g., see U.S. Pat. No.6,063,768; U.S. Pat. No. 5,670,484; and German Patent Publication DE 19852 981). German Patent Publication DE 198 52 981 discloses a compositioncontaining botulinum toxin type A and a 50% dimethyl sulphoxide (DMSO)solution for the treatment of hyperhydrosis. Although DE 198 52 981discusses that botulinum toxin may be used to treat hyperhydrosis bybeing topically applied to the skin, it is unclear whether the botulinumtoxin permeated through the epidermis of the person, or if the effectswere mediated by botulinum toxin passing through pores of the sweatglands. In any case, although DE 198 52 981 discloses that topicaladministration of botulinum toxin in a DMSO solution can be used totreat hyperhydrosis, compositions containing DMSO are not desirablebecause DMSO can irritate the skin. In addition, although U.S. Pat. No.5,670,484 discloses topical application of botulinum toxin to treat skinlesions, it does not disclose a composition containing botulinum toxinand an enhancing agent, as described herein. Furthermore, U.S. Pat. No.5,670,484 only discloses that topical administration of botulinum toxinmay inhibit cell proliferation. It is silent to topical application ofbotulinum toxin to treat disorders associated with neurosecretion ofintracellular molecules. See also WO 00/15245 and Grusser Von O-J., Dieersten systematischen Beschreibungen und tierexperimentellenUntersuchungen des Botulismus, Sudhoffa Archiv (1986), 70(2), 167-186.

Transdermal Delivery

Human skin comprises the dermis and the epidermis. The epidermis hasseveral layers of tissue, namely, stratum corneum, stratum lucidum,stratum granulosum, stratum spinosum, and stratum basale (identified inorder from the outer surface of the skin inward). The stratum corneumpresents the most significant hurdle in transdermal delivery ofmedications. The stratum corneum is typically about 10-15 μm thick, andit consists of flattened, keratised cells (corneocytes) arranged inseveral layers. The intercellular space between the corneocytes isfilled with lipidic structures, and may play an important role in thepermeation of substances through skin (Bauerova et al., Chemicalenhancers for transdermal drug transport, European Journal of DrugMetabolism and Pharmacokinetics, 2001, 26(1/2); 85-94). The rest of theepidermis below the stratum corneum is approximately 150 μm thick. Thedermis is about 1-2 mm thick and is located below the epidermis. Thedermis is innervated by various capillaries as well as neuronalprocesses.

Transdermal administration of pharmaceuticals has been the subject ofresearch in attempt to provide an alternative route of administration ofmedications without undesirable consequences associated with injectionsand oral delivery. For example, needles often cause localized pain, andpotentially exposes patients receiving injections to blood bornediseases. Oral administration suffers from poor bioavailability ofmedications due to the extremely acidic environment of the patientsstomach. Transdermal administration techniques attempt to overcome theseshortcomings by providing non-invasive administration ofpharmaceuticals. It is desirable with transdermal administration toreduce damage to a patients skin. Thus, transdermal administration ofmedication may reduce or eliminate pain associated with injections,reduce the likelihood of blood contamination, and improve thebioavailability of drugs once they are incorporated systemically.

Attempts at transdermal administration of medication have attempted toimprove the permeability of the stratum corneum. Most attempts oftransdermal therapy are directed at administering pharmaceutical agentsthat are incorporated into a patients circulatory system, and thus aresystemically administered through the skin. Some attempts have includedusing chemical enhancing agents that increase the permeability ofmolecules through the skin. Some attempts have included using mechanicalapparatus to bypass or ablate portions of the stratum corneum. Inaddition, attempts have included use of ultrasound or iontophoresis tofacilitate the permeation of pharmaceuticals through the skin. Asindicated above, the goal of these therapeutic methods is to deliver apharmaceutical agent, typically a small molecule, through the skin sothat an agent may pass to the capillary bed in the dermis where theagent may be systemically incorporated into the patient to achieve atherapeutic effect.

Although small molecules have been a major focus of transdermaladministration techniques, it is important to note that it appears thatlarge molecules, such as polypeptides, and protein complexes, are alsoamenable to transdermal administration. Erythropoietin, which is about48 kD, has also been successfully transdermally administered (Mitragotriet al., Ultrasound-mediated transdermal protein delivery, Science, 1995,269: 850-853; U.S. Pat. Nos. 5,814,599; and 6,002,961).

What is needed therefore are pharmaceutical compositions or formulationscontaining therapeutically effective amounts of neurotoxins which enablethe neurotoxin to permeate the skin of a patient and retain theneurotoxin's bioactivity to cause a therapeutic effect withoutundesirable pain associated with the administration of the neurotoxin.

SUMMARY

The present invention addresses this need and provides pharmaceuticalcompositions comprising a neurotoxin, which are able to be transdermallyadministered. The compositions of the present invention may be used todeliver the neurotoxin to a subdermal structure, such as an subdermalmuscle, a subdermal sweat gland, or a subdermal sensory neuron. Thus,the composition disclosed herein may be used to effectively treatneuromuscular disorders associated with spastic muscles, treatsympathetic neuronal disorders, such as disorders associated withhyperactive sweat glands, or to reduce inflammation or pain associatedwith inflammation, and thus, the neurotoxin may be used as an analgesic.

The following definitions apply herein:

“About” means approximately or nearly and in the context of a numericalvalue or range set forth herein means ±10% of the numerical value orrange recited or claimed.

“Local administration” means direct administration of a pharmaceuticalat or to the vicinity of a site on or within an animal body, at whichsite a biological effect of the pharmaceutical is desired. Localadministration excludes systemic routes of administration, such asintravenous or oral administration. Topical administration is a type oflocal administration in which a pharmaceutical agent is applied to aperson's skin. Topical administration of a neurotoxin, such as botulinumtoxin, excludes systemic administration of the neurotoxin. In otherwords, and unlike conventional therapeutic transdermal methods, topicaladministration of botulinum toxin does not result in significantamounts, such as the majority of, the neurotoxin passing into thecirculatory system of the patient.

“Neurotoxin” means a biologically active molecule with a specificaffinity for a neuronal cell surface receptor. Neurotoxin includesClostridial toxins both as pure toxin and as complexed with one to morenon-toxin, toxin associated proteins.

“Stabilized botulinum toxin” means a botulinum toxin that is stillbiologically active or that still is capable of binding to a target cellso that the botulinum toxin can effectively reduce or prevent exocytosisof intracellular molecules, such as neurotransmitters or peptides, fromthe cell to which the botulinum toxin is bound. Stabilized botulinumtoxins are not cytotoxic.

“Enhancing agent” refers to an agent that enhances the permeability of apatient's skin so that botulinum toxin can be absorbed by the skin toachieve a therapeutic effect. In reference to the disclosure herein,enhancing agent specifically excludes dimethylsulfoxide (DMSO) or acombination of pluronic lecithin organizer (PLO) and DMSO. An enhancingagent may include, and is not limited to, alcohols, such as short chainalcohols, long chain alcohols, or polyalcohols; amines and amides, suchas urea, amino acids or their esters, amides, AZONE®, derivatives ofAZONE®, pyrrolidones, or derivatives of pyrrolidones; terpenes andderivatives of terpenes; fatty acids and their esters; macrocycliccompounds; tensides; or sulfoxides other than dimethylsulfoxide, suchas, decylmethylsulfoxide; liposomes; transfersomes; lecithin vesicles;ethosomes; water; surfactants, such as anionic, cationic, and nonionicsurfactants; polyols; and essential oils.

A suitable neurotoxin used in the pharmaceutical compositions disclosedherein may be a neurotoxin made by a bacterium, for example, theneurotoxin may be made from a Clostridium botulinum, Clostridiumbutyricum, or Costridium beratti. In certain embodiments of theinvention, the composition may contain botulinum toxin, which may be abotulinum toxin type A, type B, type C₁, type D, type E, type F, or typeG. The botulinum toxin is present in the composition in an amount thatresults in between about 10⁻³ U/kg and about 10 U/kg of botulinum toxinpermeating through the skin. The composition may contain an amount ofbotulinum toxin that causes a therapeutic effect to persist for betweenabout 1 month and 5 years.

Other neurotoxins include recombinantly produced neurotoxins, such asbotulinum toxins produced by E. coli. In addition or alternatively, theneurotoxin can be a modified neurotoxin, that is a neurotoxin which hasat least one of its amino acids deleted, modified or replaced, ascompared to a native or the modified neurotoxin can be a recombinantproduced neurotoxin or a derivative or fragment thereof. The neurotoxinsare still able to inhibit neurotransmitter release.

The composition containing a neurotoxin, as disclosed herein, istopically administered to a patient. Because the neurotoxin is topicallyadministered the composition is preferably applied at or near a sitethat is painful or is moist from sweating. For example, if a spasticmuscle is causing pain, the composition may be applied to the skin abovethe spastic muscle to chemodenervate the underlying spastic muscle. Or,if a particular site is inflamed, such as caused by neuronal release ofsubstance P or calcitonin gene related peptide (CGRP), the compositionmay be administered at the inflammation site. In addition, if a person'ssweat glands are excessively secreting fluid, the composition may beapplied in proximity of the sweaty area to reduce the neuronalinnervation of the sweat glands. For example, the composition may beapplied to one or more arm pits, palms, or any other sweaty structure.

I have surprisingly found that a botulinum toxin, such as botulinumtoxin type A, can be transdermally administered to alleviate disordersexperienced by a human patient. The botulinum toxin used is administeredin an amount so that between about 10⁻³ U/kg and 10 U/kg pass through apatient's skin. Preferably, the botulinum toxin is present in an amountso that between about 10⁻² U/kg and about 1 U/kg are transdermally passthrough the patients skin. More preferably, the botulinum toxin ispresent in an amount so that between about 10⁻¹ U/kg and about 1 U/kgpass through the patient's skin. Most preferably, the botulinum toxin ispresent in an amount so that between about 0.1 unit and about 5 unitspass through the patient's skin to a subdermal target. Significantly,the therapeutic effects of the toxin in the composition can persist forbetween about 2 months to about 6 months when administration is ofaqueous solution of the neurotoxin, and for up to about five years whenthe neurotoxin is administered in a composition that retains the toxinand slowly releases the toxin after it has passed through the skin. Seee.g. U.S. Pat. No. 6,312,708.

Advantageously, I have discovered that by topically applyingcompositions containing botulinum toxin, potential complications, suchas systemic toxicity or botulism poisoning, are avoided even uponadministration of relatively high dosages since the stratum corneum ofthe skin still retains some impermeability. Thus, dosages of botulinumtoxin (including types A, B, C, D, E, F, or G) can range from as low asabout: 1 unit to as high as about 20,000 units, without fear of adverseside effects that may threaten the patient. The particular dosages mayvary depending on the condition being treated, and the particularenhancing agent and therapeutic regime being utilized. For example,treatment of subdermal, hyperactive muscles may require high dosages(e.g., 1000 units to 20,000 units) of botulinum toxin topically appliedin a composition containing an enhancing agent. In comparison, treatmentof neurogenic inflammation or hyperactive sweat glands may requirerelatively small topical dosages (e.g. about 1 unit to about 1,000units) of botulinum toxin.

An embodiment of the present invention can be a pharmaceuticalcomposition comprising a stabilized botulinum toxin and at least oneenhancing agent for facilitating transdermal delivery of the botulinumtoxin into a human patient by enhancing the permeability of the patientsskin. The botulinum toxin can be selected from the group consisting ofbotulinum toxin types A, B, C₁, D, E, F and G, a pure or purified (i.e.about 150 kD) botulinum toxin, as well as a native or recombinantly madebotulinum toxin. The composition can comprise between about 1 units toabout 20,000 units of the botulinum toxin, and the composition cancomprises an amount of botulinum toxin sufficient to achieve atherapeutic effect lasting between 1 month and 5 years.

Notably, the enhancing agent can be an alcohol, such as a polyalcohol.Alternately, the enhancing agent can comprise a transfersome.Additionally, the composition can comprise a plurality of enhancingagents.

A detailed embodiment of the present invention can comprise apharmaceutical composition in a transdermal patch, including astabilized botulinum toxin that permeates through a human patient's skinwithout permeating in significant amount through a blood vessel when thebotulinum toxin interacts with an enhancing agent provided in thetransdermal patch to cause a therapeutic effect of a disorder associatedwith exocytosis of a molecule from a cell. “Without permeating insignificant amount” means that less than 25% and preferably less than 5%of the botulinum toxin present in the pharmaceutical compositionpermeates into a blood vessel upon application of the transdermal patch

The botulinum toxin in the composition can be provided in a dry state inthe transdermal patch before the patch is applied to the patient's skin.The botulinum toxin can be mixed with the enhancing agent after thetransdermal patch is applied to the patient's skin. Thus, the botulinumtoxin can mix with an enhancing agent that is applied to the patient'sskin before the transdermal patch is applied to the patient's skin.

A further embodiment of the present invention includes a transdermalpatch, comprising a pharmaceutical composition, which comprises astabilized botulinum toxin; and an enhancing agent that facilitatestransdermal administration of the botulinum toxin in a bioactive form toa subdermal target site of a human patient without being administered tothe patient's circulatory system; and an adhesive disposed on one sideof the transdermal patch to removably secure the patch to the patient'sskin. The adhesive can be is disposed around a depot containing thepharmaceutical composition.

The transdermal patch can further comprise a plurality of needlesextending from one side of the patch that is applied to the skin,wherein the needles extend from the patch to project through the stratumcorneum of the skin without rupturing a blood vessel. The botulinumtoxin can be provided in a depot in the patch so that pressure appliedto the patch causes botulinum toxin to be directed through the needlesand under the stratum corneum. Furthermore, the botulinum toxin can beprovided in a dry state in a plurality of wells, each of the wellscovered by a membrane that is dissolvable with a fluid, and wherein theenhancing agent mixes with the botulinum toxin as the membrane over awell dissolves so that the absorption of the botulinum toxin isenhanced.

The present invention also encompasses a method of reducingneurotransmitter release in a subdermal structure of a patient, themethod comprising the steps of non-chemically disrupting the stratumcorneum of the patient's skin to reduce impermeability of the stratumcorneum; and applying botulinum toxin to the skin of the patient in anarea that has had the stratum corneum disrupted in the first step. Thestratum corneum can be disrupted by abrasively removing the stratumcorneum. Thus, the stratum corneum can be disrupted by applying anadhesive material to the patient's skin, and removing the adhesivematerial applied thereto. Alternately, the stratum corneum can bedisrupted by applying ultrasound at a frequency between 20 kHz and lessthan 10 MHz at an intensity that does not permanently damage thepatient's skin. Or the stratum corneum can be disrupted by passingelectrical current from a first point on the patient's skin to a secondpoint on the patient's skin. The electrical current can be passed tocreate a plurality of pores in the stratum corneum to enhance passage ofbotulinum toxin to the subdermal structures. And the botulinum toxin canbe applied in a pharmaceutical composition comprising an enhancing agentfor enhancing the delivery of the botulinum toxin through the skin.Thus, the botulinum toxin can be is incorporated into a transfersome.

The present invention also encompasses a method of relieving pain in apatient caused by a spastic muscle, the method comprising the steps of(a) applying ultrasound at a frequency between about 10 kHz and 1 MHz tothe patients skin overlying the spastic muscle; and (b) applyingbotulinum toxin to the patients skin that has received the ultrasound instep (a). Thus method can further comprise a step of abrasively removingportions of the stratum corneum of the patients skin that received theultrasound.

DESCRIPTION

The pharmaceutical composition of the present invention is capable ofdelivering a botulinum toxin, such as a purified 150 kD botulinum toxinmolecule or as a 300-900 kD botulinum toxin complex, through a person'sskin. The pharmaceutical composition contains an enhancing agent thatfacilitates the permeation of the botulinum toxin through the patient'sskin. The pharmaceutical composition is suitable for topicaladministration so that the composition may penetrate the skin andtransdermally denervate an underlying target structure, such as astructure innervated by a neuron. The composition may be a component ofa patch that may be adhesively secured to the skin so that the toxin canpass from the patch to and through the skin to denervate an underlyingtarget.

The present invention is based on the discovery that pharmaceuticalcompositions containing botulinum toxin and an enhancing agent cansuccessfully treat several types of disorders associated withneurotransmitter release when applied to a person's skin. Examples ofdisorders amenable to treatment by the topical administration of thecompositions set forth herein include, and are not limited to, wrinkles,such as brow furrows, headaches, such as migraine, headache pain,cervical dystonia, focal hand dystonia, neurogenic inflammation,hyperhydrosis, blepharospasm, strabismus, hemifacial spasm, eyeliddisorder, cerebral palsy, focal spasticity, limb spasticity, tics,tremors, bruxism, anal fissure, fibromyalgia, dysphagia, lacrimation,and pain from muscle spasms. The topical administration of the toxinreduces the pain experienced by the patient when the toxin isadministered because the patient does not need to be stuck with a needlethat activates sensory pain neurons below the skin. The compositionsdisclosed herein provide localized relief with a botulinum toxin,without risking systemic administration of the botulinum toxin.

The neurotoxins used in accordance with the invention disclosed hereinare neurotoxins that inhibit transmission of chemical or electricalsignals. The neurotoxins preferably are not cytotoxic to the cells thatare exposed to the neurotoxin. The neurotoxin may inhibitneurotransmission by reducing or preventing exocytosis ofneurotransmitter from the neurons exposed to the neurotoxin. Thesuppressive effects provided by the neurotoxin should persist for arelatively long period of time, for example, for more than two months,and potentially for several years.

Examples of neurotoxins used in the compositions, include, and are notlimited to, neurotoxins made from: Clostridium bacteria, such asClostridium botulinum, Clostridium butyricum and Clostridium beratti. Inaddition, the neurotoxins used in the methods of the invention may be abotulinum toxin selected from a group of botulinum toxin types A, B, C,D, E, F, and G. In one embodiment of the invention, the neurotoxinadministered to the patient is botulinum toxin type A. Botulinum toxintype A is desirable due to its high potency in humans, readyavailability, and known use for the treatment of skeletal and smoothmuscle disorders when locally administered by intramuscular injection.The present invention also includes the use of (a) neurotoxins obtainedor processed by bacterial culturing, toxin extraction, concentration,preservation, freeze drying, and/or reconstitution; and/or (b) modifiedor recombinant neurotoxins, that is neurotoxins that have had one ormore amino acids or amino acid sequences deliberately deleted, modifiedor replaced by known chemical/biochemical amino acid modificationprocedures or by use of known host cell/recombinant vector recombinanttechnologies, as well as derivatives or fragments of neurotoxins somade. These neurotoxin variants should retain the ability to inhibitneurotransmission between or among neurons, and some of these variantsmay provide increased durations of inhibitory effects as compared tonative neurotoxins, or may provide-enhanced binding specificity to theneurons exposed to the neurotoxins. These neurotoxin variants may beselected by screening the variants using conventional assays to identifyneurotoxins that have the desired physiological effects of inhibitingneurotransmission.

Botulinum toxins for use according to the present invention can bestored in lyophilized, vacuum dried form in containers under vacuumpressure or as stable liquids. Prior to lyophilization the botulinumtoxin can be combined with pharmaceutically acceptable excipients,stabilizers and/or carriers, such as albumin. The lyophilized materialcan be reconstituted with saline or water to create a solution orcomposition containing the botulinum toxin to be administered to thepatient.

Although the composition may only contain a single type of neurotoxin,such as botulinum toxin type A, as the active ingredient to suppressneurotransmission, other therapeutic compositions may include two ormore types of neurotoxins, which may provide enhanced therapeuticeffects of the disorders. For example, a composition administered to apatient may include botulinum toxin type A and botulinum toxin type B.Administering a single composition containing two different neurotoxinsmay permit the effective concentration of each of the neurotoxins to belower than if a single neurotoxin is administered to the patient whilestill achieving the desired therapeutic effects.

An enhancing agent used in combination with the neurotoxin in thepharmaceutical composition may be any non-DMSO based enhancing agentthat enhances the permeability of the skin so that bioactive neurotoxinmay act at a desired target structure. The enhancing agent preferablydoes not injure the skin, and more preferably, temporarily permeabilizesthe skin so that once the neurotoxin has been delivered through theskin, the skin reduces its permeability to other factors.

In one embodiment of the invention, the enhancing agent is an alcohol.Examples of alcohols include short chain alcohols, such as alcoholshaving between about 2-5 carbon atoms. Some short chain alcohols includeethanol, isopropanol, methanol, and isobutanol, or combinations thereof.The alcohols may be mixed in the composition so that the concentrationof alcohol in the composition is between about 10% and 40%. The alcoholmay be admixed with glycerin to reduce potential irritation caused byhigher concentrations of alcohol. Long chain alcohols are also useful toenhance the transdermal administration of neurotoxins, such as botulinumtoxins. Examples of long-chain alcohols include alcohols having betweenabout 8 and 12 carbon atoms, and some specific examples includen-dodekano, klenbuterol, and albuterol. Polyalcohols may also be usedwith the neurotoxin. Examples include propylene glycol, glycerol,polyethylene glycol, and dexpantheol, and combinations thereof.

In another embodiment, an enhancing agent may be a vesicle that is ableto store the neurotoxin within the vesicle. The vesicle can diffusethrough the skin and thereby deliver the neurotoxin to the target site.The vesicle may be a lipid vesicle. In one specific embodiment, theneurotoxin is incorporated into a transfersome, which are deformablecarries containing lipids and membrane softeners (e.g., Hofer et al.,New Ultradeformable Drug Carriers for Potential Transdermal Applicationof Interleukin-2 and Interferon-α: Theoretic and Practical Aspects,World J. Surg. 24, 1187-1189 (2000); and U.S. Pat. No. 6,165,500).Surprisingly, it has been discovered that transfersomes sufficientlytransport neurotoxins including botulinum toxin complexes, across theskin to achieve a therapeutic effect. In other words, the neurotoxin isable to be delivered to a target site and still be bioactive afterdiffusing through the skin.

The compositions of the invention may be used in an application devicethat permits application of the composition to a target site on the skinwithout applying the composition to non-target site areas of the skin.For example, a device may be employed that allows the composition to beapplied without first applying the composition to one's fingers, whichmay lead to undesirable paralysis of the fingers. Suitable devicesinclude spatulas, swabs, syringes without needles, and adhesive patches.Use of spatulas or swabs, or the like may require the device to beinserted into a container containing the composition. Using syringes oradhesive patches may be accomplished by filling the syringe or patchwith the composition. The composition may then be topically spread bythe spatulas or swabs, or may be expelled from the syringes onto theperson's skin.

In one embodiment of the invention, the composition containing theneurotoxin and the enhancing agent is provided in an adhesive patch.Some examples of adhesive patches are well known. For example, see U.S.Pat. Nos. Des. 296,006; 6,010,715; 5,591,767; 5,008,110; 5,683,712;5,948,433; and 5,965,154. Transdermal patches are generallycharacterized as having an adhesive layer, which will be applied to aperson's skin, a depot or reservoir for holding a pharmaceutical agent,and an exterior surface that prevents leakage of the pharmaceutical fromthe depot. The exterior surface of a patch is typically non-adhesive.

In accordance with the present invention, the neurotoxin is incorporatedinto the patch so that the neurotoxin remains stable for extendedperiods of time. The neurotoxin may be incorporated into a polymericmatrix that stabilizes the neurotoxin, and permits the neurotoxin todiffuse from the matrix and the patch. The neurotoxin may also beincorporated into the adhesive layer of the patch so that once the patchis applied to the skin, the neurotoxin may diffuse through the skin. Inaccordance with such an embodiment, the adhesive preferably comprises anenhancing agent, as disclosed herein. In one embodiment, the adhesivelayer may be heat activated where temperatures of about 37 degreesCelsius cause the adhesive to slowly liquefy so that the neurotoxindiffuses through the skin. The adhesive may remain tacky when stored atless than 37 degrees Celsius, and once applied to the skin, the adhesiveloses its tackiness as it liquefies. The administration of the toxin iscomplete once the patch no longer adheres to the skin.

Alternatively, the neurotoxin may be provided in one or more wells orpockets disposed near the surface of the patch that will contact theskin. In one embodiment, the neurotoxin is stored in the wells in adried, or lyophilized state. Storing such patches in a cooled atmosphere(e.g., about 4 degrees Celsius) maintains the stability the neurotoxin.A patch may be removed from the cool atmosphere when needed, and appliedto a person's skin where the neurotoxin may be solubilized upon themixing with fluid, such as water or saline. The fluid may be providedseparately or as a component of the patch. For example, fluid may beprovided on a person's skin so that when the patch containing the driedneurotoxin interacts with the fluid, the neurotoxin is exposed to thefluid and is solubilized. The solubilized neurotoxin may then be able tobe absorbed by the skin. As another example, the patch may contain oneor more wells or pockets to hold fluid in the patch. The fluid may beforced from the wells or pockets to cause the fluid to mix with thedried neurotoxin. In such embodiments, the enhancing agent may beprovided in the fluid to enhance the permeability of the skin to theneurotoxin. For example, the fluid may be provided in a pocket in thepatch. Pressure exerted on the patch causes the pocket to rupture andrelease the fluid so that it mixes with the dried neurotoxin. Thecomposition containing the neurotoxin may thus diffuse through thepatient's skin. As another example, fluid, including gels and creamscontaining water may be applied to the skin at a target site. The patchcontaining the dried neurotoxin may then be applied to the skin wherethe fluid mixes with the neurotoxin and the composition diffuses intothe skin.

In patches containing wells of dried neurotoxin, it is desirable to sealthe wells so that the neurotoxin remains in the wells until theneurotoxin is to be administered. Accordingly, the wells are sealed witha membrane or film that prevents the neurotoxin from diffusing from thewells in the neurotoxin's dry state, but that permits the neurotoxin todiffuse from the wells when it is solubilized. The membrane may eitherbe porous or nonporous. In one embodiment, the membrane comprisescellulose or starch, and more particularly, the membrane may containpolyvinyl alcohol, polyethylene oxide, and hydroxypropyl methylcellulose. The membrane is thin (ranging in thickness from about 1 μm toabout 1 mm) and dissolves upon contacting fluid. Thus, fluid placed onthe person's skin or fluid directed from a pocket in the patch maycontact the cellulose membrane and cause the membrane to dissolve. Afterdissolving, the fluid mixes with the dried neurotoxin and solubilizesthe neurotoxin. The composition then diffuses through the patients skin.

Additionally, the transdermal patch may include a plurality of smallneedles that extend through the stratum corneum, but do not extend intothe dermis to rupture blood vessels. The needles may be between 20 μmand 1 mm long when extending from the dermal surface of the patch. Thus,the needles extend through the stratum corneum, but terminate before thedermis where the capillary beds are located. The needles may be solid orhollow. Hollow needles may have a lumen extending along their length sothat the composition can pass from the depot in the patch to the end ofthe needle in the epidermis. Solid needles may be used to permit thecomposition to diffuse along the outer surface of the needle into theepidermis. Surprisingly, it has been discovered that this length ofneedles is optimal to reduce potential pain caused by longer needlesactivating sensory pain fibers. Thus, the composition containing theneurotoxin may be applied subdermally without significant, if any, painto the patient.

Accordingly methods of inhibiting neurotransmitter release in subdermalstructures may include steps of disrupting the stratum corneum to reducethe impermeability of the stratum corneum, and applying a botulinumtoxin to the skin location in which the stratum corneum has beendisrupted. Disrupting the stratum corneum refers to either completelyremoving the stratum corneum from a region of a patient's skin, orpartially removing portions of the stratum corneum at a location on thepatient's skin so that relatively small stratum corneum-free regions ofskin are present. The skin may be disrupted using any suitable methodwithout imparting significant pain to the patient. In preferredembodiments of the methods, the stratum corneum is non-chemicallydisrupted. For example, the stratum corneum may be abrasively scrubbedto disrupt the laminar barrier of the stratum corneum. Or, the stratumcorneum may be disrupted by applying an adhesive, such as adhesive tapeor wax, to the skin, and subsequently removing the adhesive from theskin. Because such methods of disrupting the stratum corneum may causesome pain, it may be desirable to provide a topical anesthetic to theskin, such as lidocaine cream, to temporarily reduce any pain that maybe caused by the disruption.

Additional transdermal methods that non-chemically enhance the skin'spermeability include low frequency ultrasound (20 kHz to 1 MHz).Ultrasound is defined as sound at a frequency of between about 20 kHzand 10 MHz, with intensities of between 0 and 3 W/cm². Low frequencyultrasound, as used herein, refers to ultrasound at a frequency that isless than 1 MHz, and preferably in the range of 20 kHz to 40 kHz. Theultrasound is delivered in pulses, for example, 100 msec pulses at afrequency of 1 Hz. The intensity of the ultrasound may vary between 0and 1 W/cm², and frequently varies between 12.5 mW/cm² and 225 mW/cm².Typical duration of exposure to ultrasound is between about 1 and about10 minutes. The ultrasound is applied without causing an increase inskin temperature greater than about 1 degree Celsius. Low frequencyultrasound may be used alone or in combination with the composition toimprove the permeability of the skin to the neurotoxin. Examples ofultrasound techniques for improving skin permeability may be found inU.S. Pat. Nos. 6,002,961 and 5,814,599. Surprisingly, it has beendiscovered that low frequency ultrasound, when applied in conjunctionwith a composition containing a botulinum toxin, permeabilizes the skinbut does not substantially alter the three dimensional conformation ofthe neurotoxin, such as purified botulinum toxin or botulinum toxincomplexes. Thus, the bioactivity of the neurotoxin is maintained and thedisorder is substantially treated.

Additionally, the ultrasound may be delivered prior to application ofthe botulinum toxin to the skin. It has been discovered that lowfrequency ultrasound when applied before the topical application ofbotulinum toxin, temporarily disrupts' the stratum corneum so thatsubsequent topical application of botulinum toxin achieves a therapeuticeffect. In other words, the disruption caused by the ultrasound persistsfor several minutes, for example between about 10 and 30 minutes, toprovide relatively easy transdermal delivery of botulinum toxin to thepatient. After about 30 minutes, the stratum corneum begins to resumeits natural structure, and the permeability of the stratum corneumtemporally decreases. Thus, one method of the invention, includes thestep of applying low frequency ultrasound, to one or more regions of theskin, and subsequently topically applying botulinum toxin to thoseregions of the skin that were exposed to the low frequency ultrasound,where the botulinum toxin is provided in a composition containing anenhancing agent, which facilitates prolonged penetration of thebotulinum toxin to the patient.

Additional approaches include iontophoresis which can help deliver thebotulinum toxin to a subdermal target site by passing electrical currentacross a patch containing a composition comprising botulinum toxin. Inone embodiment, an electrode may be applied on the external surface ofthe transdermal patch, and a ground electrode is provided elsewhere on apatient's skin. Small direct current is applied through the electrodepositioned on the transdermal patch to urge the botulinum toxin in thecomposition through the patients skin. The amount of current istypically less than 1 mA/cm², and in a preferred embodiment, the currentis applied in an amount between 0.3 mA cm² and 0.7 mA/cm². Because theeffectiveness of transdermal delivery of the botulinum toxin through theskin is at least partially dependent on the polarity of the botulinumtoxin, it may be desirable to increase the acidity of the composition tolower the pH of the composition and impart a charge on the botulinumtoxin to facilitate the effectiveness of the electrical current intransporting the toxin through the skin. The pH of the compositioncontaining the botulinum toxin can be lowered to as low as 4 withoutsignificantly compromising the bioactivity of the molecule; however,preferred pH ranges are between 5.5 and 7.2. Additionally, the currentis passed through the electrodes for a time that does not permanentlydamage (e.g., burn) the skin. For example, the current may be passed fora period of time between about 1 minute and 15 minutes. For longerapplications, it is desirable to pulse the current to reduce potentiallydamaging effects caused by the electricity.

The neurotoxin may be topically administered by any suitable method asdetermined by the attending physician. The methods of administrationpermit the neurotoxin to be administered locally to a selected targettissue. Methods of administration include coating the skin with thecomposition so that the composition covers at least a portion of thetarget site. Administration methods also include applying a transdermalpatch to the target site of the skin and causing the neurotoxin in thetransdermal patch to diffuse into the skin. For extended applications,adhesive patches are utilized so that the composition can slowly diffuseinto the skin without repeated applications of the patch. For example, apatch may include a microprocessor that provides periodic release ofneurotoxin from the patch. Microprocessor patches may be especiallyadvantageous in patches that have the microneedles or low frequencyultrasound devices, as discussed above. The microprocessor can provide atimed release of the composition depending on the particular conditionbeing treated. An example of a microprocessor controlled pharmaceuticaltreatment device may be found in U.S. Pat. No. 6,334,856.

Diffusion of biological activity of a botulinum toxin within a tissueappears to be a function of dose and can be graduated. Jankovic J., etal Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page 150.Thus, diffusion of botulinum toxin can be controlled to reducepotentially undesirable side effects that may affect the patientsdisorder. For example, the neurotoxin may be administered so that theneurotoxin primarily effects sensory neurons involved in inflammation,and does not affect other subdermal targets. In such a case, thecomposition may be applied for a relatively short period of time (e.g.,1-4 hours) to permit only local diffusion into the dermis of the patientwhere the sensory neurons terminate. The neurotoxin may thus act on thesensory neurons to decrease the release of substance P or CGRP to reduceinflammation and pain associated with inflammation.

Without wishing to be bound by any particular theory, a mechanism can beproposed for the therapeutic effects achieved with the compositionpracticed according to the present invention. The enhancing agentsdisclosed herein appear to solubilize the stratum corneum of theepidermis and increase the fluidity of the neurotoxin thus maintainingthe bioactivity of the neurotoxin when it reaches the subdermal target.Importantly, the compositions and methods herein of topicallyadministering the neurotoxin permit the neurotoxin to be administered toa patient without resulting in systemic toxicity. As indicated herein,prior art approaches of transdermally administering non-botulinum toxintherapeutic agents have addressed systemic administration of thetherapeutic agents via the skin. In additional prior art approaches fortopically applying botulinum toxin to patients have not included the useof enhancing agents, as disclosed herein.

As set forth above, I have discovered that compositions containing aneurotoxin and an enhancing agent surprisingly provides effective andlong lasting treatment of disorders associated with neuronal activitynear a patient's skin, and reduces the symptoms associated with thedisorder. In its most preferred embodiment, the present invention ispracticed by topical administration of botulinum toxin type A.

EXAMPLES

The following examples set forth specific compositions and methodsencompassed by the present invention to treat patients, and are notintended to limit the scope of the invention. For example, although thefollowing examples are directed to compositions containing botulinumtoxin type A, I have discovered that botulinum toxin types B, C, D, E,F, and G are equally effective in being transdermally administered inthe compositions set forth herein. It is noted that the dosages of theparticular type of botulinum toxin may be adjusted as needed from theparticular dosages disclosed herein, as understood by persons ofordinary skill in the art. As indicated above, the transdermaladministration methods disclosed herein permit a relatively broad rangeof concentrations of botulinum toxin without risking the patientshealth.

Example 1

One hundred units of botulinum toxin type A are dissolved in 1 mL ofwater are mixed with 1 mL of 90% ethanol and 1 mL of polyethyleneglycol. A syringe is filled with the composition of the botulinum toxin.The viscous solution is expelled from the syringe onto a patient's palmwho is complaining of sweaty palms. The solution is spread with aspatula over the entire palmar surface. The patient's hand is coveredwith a plastic bag with an airtight seal for about one hour to reducethe rate of evaporation of the composition. The bag is removed and thepatient washes his hand. Approximately 2 days later, the patient noticesthat the hand receiving treatment no longer is sweaty while theuntreated hand remains sweaty. The reduction in sweat is maintained forabout 6 weeks, and then gradually returns. The procedure is repeated forboth hands, and both hands show a marked reduction in sweating after aperiod of about 2 days.

Example 2

A 1 mL 10% suspension of transfersomes is made from 85.8 mg naturalphosphatidyl choline and 14.2 mg sodium cholate. Approximately 0.9 mL ofphosphate buffer is added to solubilize the lipids. The suspension isfiltered several times to achieve a suspension of approximately uniformsized vesicles. Approximately 1000 units of botulinum toxin type A(BOTOX®) are added to the vesicles and are stored at 4 degrees Celsiusfor at least two days and up to about 30 days.

A patient with brow furrows requests botulinum toxin to reduce thewrinkles. The patient is asked to lay down. A suspension of BOTOX® andtransfersomes as described above is topically applied to the patient'sforehead. After about 1 hour, the suspension has evaporated. The patientis instructed to wash his face approximately 6 hours later. In about 2-3days, the patient begins to notice that the forehead wrinkles arereduced in number. At about 7 days, the wrinkles are gone. The effectsof the BOTOX® last for about 4 months.

Example 3

Lyophilized BOTOX® is provided in a plurality of wells located on adermal side of a transdermal adhesive patch. The transdermal patch hasdimensions of approximately two inches by three inches (5 cm×7.5 cm).The wells are organized in grids of approximately 1 cm² on the dermalside of the patch (i.e., the side of the patch that will be adjacent theskin). Each grid contains approximately 100 wells. The dermal side ofthe patch is fabricated from polyethylene terephthalate (PET). Each wellcontains between about 50-100 units of lyophilized BOTOX. The wells aresealed with a dissolvable membrane film made of polyvinyl alcohol,polyethylene oxide, and hydroxypropyl methyl cellulose. An adhesiveborder is provided around the grid. The adhesive border is approximately1 cm wide and comprises a rubber adhesive, such as R-1072 from B.F.Goodrich Co. A patch containing the lyophilized BOTOX in a plurality ofwells may be stored at 4 degrees Celsius for several months withoutaffecting the bioactivity of the toxin.

Two patches, as described above, are applied to the skin of a patient'slower back on either side of the spinal cord at a location demonstratingextreme muscle hyperactivity. Prior to application, the skin is preparedby cleaning the site with 95% ethanol. A gel containing water, 80%ethanol, and polyethylene glycol is applied in an area about one inch bytwo inches on either side of the spinal cord. Each patch is adhesivelyapplied to the back where the gel is located. The patch is left in placefor 5-7 days. The gel dissolves the membrane and solubilizes thebotulinum toxin in the wells. After about 4 days, the patient noticesrelaxation of his lower back muscles, and a reduction in pain associatedwith the muscle contractions. By 7 days, the patient indicates the painis completely gone. The pain relieving effect persists for about 5months.

Example 4

A patient with cervical dystonia receives four transdermal patches, eachhaving dimensions of approximately 2 inches by 3 inches, applied to theskin overlying the rigid muscles. The transdermal patches contain adepot of dried BOTOX®, and a pocket of saline. An ultrasound device isapplied over the patch. Ultrasound is applied to the patch and thepatients skin at a frequency of 15 kHz for a period of 10 minutes. Theultrasound energy is pulsed to reduce damaging the patient's skin. After10 minutes, the physician removes the ultrasound device, and appliespressure to the transdermal patches to cause the pocket of saline torupture. The saline that is expelled from the pocket mixes with thebotulinum toxin to solubilize the toxin. The composition is deliveredthrough the skin by diffusion. The patches are left in place for about 5hours. Approximately 2-3 days after the treatment, the patientexperiences some relief of pain and relaxation of the muscles.Approximately 7 days after treatment, the pain is almost completelyrelieved. The therapeutic effects persist for about 3 months.

Example 5

A patient with suffering from palmar hyperhydrosis requests botulinumtoxin therapy. The physician evaluates the patient and determines thatthe patient is a reasonable candidate for botulinum toxin therapy. Thephysician abrasively scrubs the patient's palms with a pumice stone.After the majority of the skin has been roughened, the physician appliesa saline based gel containing approximately 10 units of BOTOX® to thepatients palm. The patient's hands are kept in plastic bags that havebeen sealed around the patient's wrists to prevent rapid evaporation ofthe gel. The patient's hands are left in the bags for about 4 hours.About 2 days after treatment, the patient notices a reduction in thehyperhydrosis of his palms. By 7 days, the sweating is completelyeliminated, and the patient does not report any appreciable loss ofmuscle activity. The hyperhydrosis alleviating effects persist for aboutsix to eight weeks.

Example 6

A transdermal patch containing approximately 1000 units of BOTOX® isapplied to a patient's inflammed elbow after the skin of the elbow hasbeen prepared by abrasively scrubbing the skin with a pumice stone.After scrubbing the skin, a gel is applied to the elbow before thetransdermal patch is applied. The patch is applied to the elbow in aflexed position. The gel dissolves the cellulose membrane of the patchand solubilizes the botulinum toxin contained therein. About 1 hourafter the patch is applied to the elbow, enough time for the membrane todissolve and the toxin to be solubilized, an electrode is placed on theouter surface of the patch. A ground electrode is attached to thepatient's torso. Current is passed through the electrode at an intensityof 0.5 mA/cm² for 5 minutes. After resting for 2 minutes current isagain passed through the electrode for 5 minutes. The patient leaves thephysician's office, and is asked to leave the patch in place for about 4days. After about 2 days, the patient notices a reduction ofinflammation accompanied by a reduction in pain. By about 7 days, thepain is almost completely alleviated. The relief provided by thebotulinum toxin persists for about 4 months.

Transdermal compositions containing botulinum toxin and methods ofadministering such compositions according to the invention disclosedherein have many benefits and advantages, including the following:

1. the symptoms, such as the symptoms associated with hyperactiveneuronal systems associated with spastic muscles, inflammation, orhyperhydrosis can be dramatically reduced.

2. the symptoms can be reduced for from about two to about five monthsper application of neurotoxin to the skin and for from about one year toabout five years upon use of slow release compositions and patches.

3. the administered neurotoxin shows little or no tendency to diffuse orto be transported away from subdermal site.

4. few or no significant undesirable side effects occur from topicaladministration of the neurotoxin.

5. the suppressant effects of the compositions can result in thedesirable side effects of greater patient mobility, a more positiveattitude, and an improved quality of life.

6. high, therapeutic doses of a neurotoxin can be delivered to subdermaltarget tissue over a prolonged period without systemic toxicity.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe compositions and methods of the present invention.

All references, articles patents, applications and publications setforth above are incorporated herein by reference in their entireties.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

1. A pharmaceutical composition comprising: a stabilized botulinumtoxin; and at least one enhancing agent for facilitating transdermaldelivery of the botulinum toxin into a human patient by enhancing thepermeability of the patient's skin.
 2. The composition of claim 1,wherein the botulinum toxin is selected from the group consisting ofbotulinum toxin types A, B, C.sub.1, D, E, F and G.
 3. The compositionof claim 1, wherein the botulinum toxin is botulinum toxin type A. 4.The composition of claim 1, wherein the botulinum toxin is a purifiedbotulinum toxin.
 5. The composition of claim 1, wherein the compositioncomprises between about 1 units to about 20,000 units of botulinumtoxin.
 6. The composition of claim 1, wherein the composition comprisesan amount of botulinum toxin to achieve a therapeutic effect lastingbetween 1 month and 5 years.
 7. The composition of claim 1, wherein theenhancing agent is an alcohol.
 8. The composition of claim 7, whereinthe alcohol is a polyalcohol.
 9. The composition of claim 1, wherein theenhancing agent comprises a transfersome.
 10. The composition of claim1, wherein the composition comprises a plurality of enhancing agents.11. A pharmaceutical composition in a transdermal patch, thepharmaceutical composition comprising: a stabilized botulinum toxin thatpermeates through a human patient's skin without permeating insignificant amount through a blood vessel when the botulinum toxininteracts with an enhancing agent provided in the transdermal patch tocause a therapeutic effect of a disorder associated with exocytosis of amolecule from a cell.
 12. The composition of claim 11, wherein thebotulinum toxin is provided in a dry state in the transdermal patchbefore the patch is applied to the patient's skin.
 13. The compositionof claim 11, wherein the botulinum toxin is botulinum toxin type A. 14.The composition of claim 11, wherein the botulinum toxin is mixed withthe enhancing agent after the transdermal patch is applied to thepatient's skin.
 15. The composition of claim 14, wherein the botulinumtoxin mixes with an enhancing agent that is applied to the patient'sskin before the transdermal patch is applied to the patient's skin. 16.A transdermal patch, comprising a pharmaceutical composition, whichcomprises: a stabilized botulinum toxin; and an enhancing agent thatfacilitates transdermal administration of the botulinum toxin in abioactive form to a subdermal target site of a human patient withoutbeing administered to the patient's circulatory system; and an adhesivedisposed on one side of the transdermal patch to removably secure thepatch to the patient's skin.
 17. The transdermal patch of claim 16,wherein the adhesive is disposed around a depot containing thepharmaceutical composition.
 18. The transdermal patch of claim 16,further comprising a plurality of needles extending from one side of thepatch that is applied to the skin, wherein the needles extend from thepatch to project through the stratum corneum of the skin withoutrupturing a blood vessel.
 19. The transdermal patch of claim 18, whereinthe botulinum toxin is provided in a depot in the patch so that pressureapplied to the patch causes botulinum toxin to be directed through theneedles and under the stratum corneum.
 20. The transdermal patch ofclaim 16, wherein the botulinum toxin is provided in a dry state in aplurality of wells, each of the wells covered by a membrane that isdissolvable with a fluid, and wherein the enhancing agent mixes with thebotulinum toxin as the membrane over a well dissolves so that theabsorption of the botulinum toxin is enhanced.
 21. (canceled) 22.(canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)27. (canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled) 31.(canceled)
 32. (canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)